This work was funded in part by DK089016 and L30 RR030244 (MAC), CA153036 (AS), and P30-DK52574 (to the Washington University Digestive Diseases Research Core). A.I.T. was a Howard Hughes Medical Institute Medical Research Training Fellow.

1. Colitis-associated Cancer Induction 
<ol>
 <li>Set aside cages of sex and age-matched 6-8 week old mice to be used for experimental and control groups. Mice can be individually labeled with tail markings or ear clips. </li>
 <li>On day 0, record baseline weights and inject each mouse intraperitoneally (IP) with 10 mg/kg of AOM working solution (1 mg/ml in isotonic saline, diluted from 10 mg/ml stock solution in dH2O kept at -20 deg C). Based on experience, this dose can be adjusted between 7-14 mg/kg and/or repeated early in the experiment. </li>
</ol>
Caution: AOM is a volatile genotoxic agent and should be handled carefully according to the accompanying MSDS. Dilutions should be prepared in a chemical hood, maintained on ice, and discarded following institution specific protocols.
<ol start="3">
 <li>Make a 2.5% (2.5 g/100 ml) DSS solution in distilled water and pass through a 0.22 &mu;m cellulose acetate filter by vacuum. This dose can be adjusted between 1-3.5% depending on mouse strain and environment. Once prepared, DSS solution can be kept refrigerated for up to 1 week. </li>
 <li>On day 7, supply DSS solution to mice as their drinking water. Approximately 250 ml/cage will be needed every time new DSS is provided for a maximum of 5 mice/cage; however, these are only estimations and will vary depending on the type of water bottles used in your animal facility. </li>
 <li>To provide a continuous supply of DSS for seven days, DSS solution should be replaced in clean bottles three times (every 2-3 days) during this period. Some investigators measure the amount of DSS consumed prior to replacing with new solution as a measure of exposure. </li>
 <li>On day 14, switch cages back to standard drinking water for two weeks.</li>
 <li>Repeat steps (1.4) - (1.6) on days 28 and 49 to provide a second and third cycle of DSS. A DSS "cycle" consists of one week of DSS in the drinking water followed by 2 weeks of regular (autoclaved) water. </li>
</ol>
2. Clinical Assessment Colitis and Tumor Progression
<ol>
 <li>Mice should be weighed/observed 2-3 times per week. Percent weight loss relative to baseline is used as a surrogate measure of colitis severity. Regular assessments of rectal bleeding, diarrhea, or prolapse can be made depending on experimental goals and reported according to various scoring systems.9, 10 </li>
 <li>A weight loss of up to 10% along with 2-3 days of diarrhea and rectal bleeding can be expected in the week following a full cycle of DSS (daily observation may be useful during this immediate post-DSS period). </li>
</ol>
It is possible that some mice may not recover; mice losing greater than 20% of their weight are less likely to survive and may require early euthanasia. A single IP injection of 0.5 - 1.0 ml saline in such mice can be a useful supportive measure towards correcting fluid volume lost due to diarrhea.
3. Murine Endoscopy (Optional)
<ol>
 <li>Endoscopy following the second or third cycle of DSS can be performed to confirm tumor growth in vivo prior to sacrifice. As the AOM/DSS model is quite reliable in producing tumors, this step is generally not necessary. Only a few mice should evaluated using this method, as it has the potential to disrupt mouse tumors.11 </li>
 <li>Anesthetize the mouse with inhaled or injectable sedation. </li>
 <li>After mouse is anesthetized, secure the mouse to a flat board with tape at the tail and chest. </li>
 <li>Advance the murine endoscope into the mouse rectum gently using saline to inflate and irrigate to improve intraluminal view and remove fecal contents. (Note that endoscopy can also be performed using air insufflation as an alternative to saline and this technique may offer improved visualization of the tumor.12) </li>
</ol>
4. Mouse Sacrifice and Colon Harvesting
<ol>
 <li>On day 70, each AOM/DSS treated mouse should harbor multiple colon tumors and be ready for assessment. This date can be delayed weeks to months if larger tumors are desired. (Of note, mice with severe rectal prolapsed may need to be euthanized early to avoid excessive discomfort to animals as per institutional animal care committees.) </li>
 <li>Prior to necropsy, individually euthanize mice with isoflurane and cervical dislocation (or other institutionally-approved method). Final weight and other measurements can be made at this time. </li>
 <li>Lay the mouse with its ventral side exposed on a cutting board. Cover the abdomen with 70% ethanol to prevent hairs from contaminating the abdominal contents during extraction of the colon. </li>
 <li>Use forceps to grasp the midline of the abdomen and make a small incision to expose the peritoneum. </li>
 <li>Extend this incision on each side of the abdomen along the costal margin. </li>
 <li>Use scissors to cut through the pelvis so that the colon can be harvested down to the anorectal junction. This is important because DSS colitis injury is greatest at the distal rectum and correspondingly, this is the region of greatest tumor development. </li>
 <li>Additional tissues such as mesenteric lymph nodes may also be harvested at this time. </li>
</ol>
5. Preparing the Colon for Macroscopic Analysis
<ol>
 <li>Using an 18G gavage needle attached to a 5 ml syringe, flush the colon with ice-cold phosphate-buffered saline (PBS) such that fluid exits in the physiologic direction. If colon tissue will be used for RNA or protein-based analysis, it can be kept cold by employing pre-chilled work trays. </li>
 <li>Open the flushed colon longitudinally along its mesentery and paint onto the cold tray. Tumors are more easily visualized when the colon is placed on a dark surface. Alternatively, 1% Alcian blue dye can be applied to highlight tumors. Assess tumor number and size with a ruler or digital caliper. </li>
 <li>Small sections of tumor and adjacent normal tissue can be excised at this point for future analysis by RNA, protein, or immunohistochemical (IHC) methods, leaving some portion of the remaining colon for histological assessment. </li>
</ol>
Digital photography of the gross colon specimens immediately following step 5.2 can be helpful for precise analysis of tumor burden, especially if significant portions of the colons are excised for other forms of analysis as described above. Tumor burden (%) can be calculated as tumor area/total colon area using free software such as ImageJ.
6. Preparing the Colon for Histological Assessment
<ol>
 <li>Apply 10% formalin to the remaining colon on the cold tray via syringe or finger. Allowing the tissue to fix for at least 30 sec in this manner facilitates transfer to the fixation basin. Ensure residual formalin is wiped off prior to painting a new opened colon on the same tray. </li>
 <li>Transfer colons to a basin filled with 10% formalin and pin down at edges. </li>
 <li>After 3 hr of fixation, transfer colons to 70% ethanol. Colons can be preserved in 70% ethanol indefinitely. </li>
 <li>Three longitudinal sections of colon (proximal, mid, distal/rectum) can be stabilized in 2% agar and submitted for paraffin embedding. Assure that the proximal colon and distal colon/rectum are oriented in a uniform manner for each mouse. </li>
 <li>Dysplasia, crypt damage, and inflammation can be described and assessed by an experienced individual according to previously published protocols.10, 13-15 Mice can be sacrificed prior to day 70 if the experimenter is interested in detecting early dysplastic events. </li>
</ol>
7. Representative Results
The AOM/DSS model described herein allows the researcher to reliably generate colon tumors in mice. Tumor growth in this model is directly influenced by the associated inflammatory process. Colitis severity should be monitored clinically by following weight loss and presence of diarrhea/hematochezia (Figure 2). These signs of disease activity tend to begin by day 5 of the DSS cycle and for four or more days after DSS is removed. Rarely, mice with a significant rectal tumor burden can develop rectal prolapse. After the second or third DSS cycle diarrhea may become persistent. Typically tumors are present and identifiable by murine colonoscopy prior to the third cycle of DSS (Figure 3). Additional time and a third course of DSS results in larger tumors at the time of harvest (Figure 4). Use of topically applied Alcian blue stain may be used to highlight tumors (Figure 5). Photographs of colonic tumors will assist in generating tumor measurements which can be used to quantitatively compare tumor production and size between experimental groups (Figure 6). Fixed and paraffin embedded colon specimens can then be evaluated for histology or with the use of immunohistochemical staining (Figure 7 and 8).
<img src="/files/ftp_upload/4100/4100fig1.jpg" alt="Figure 1" /> 
Figure 1. Schematic of AOM and DSS administration. AOM (10 mg/kg) is injected on day 0. At the beginning of the second week (day 7), 2.5% DSS solution is administered to mice in their drinking water. Seven days of DSS is followed by two weeks of autoclaved water. An additional two cycles of DSS are administered prior to sacrifice. 
<img src="/files/ftp_upload/4100/4100fig2.jpg" alt="Figure 2" /> 
Figure 2. Mouse weight relative to baseline during AOM and DSS administration. Note that in the week following each DSS cycle, mice lose 5-10% of their body weight. Weight loss in this experiment is a surrogate marker for colitis severity. 
<img src="/files/ftp_upload/4100/4100fig3.jpg" alt="Figure 3" /> 
Figure 3. View of tumors in distal colon via murine endoscopy at day 50 of AOM/DSS treatment. Note the multiple polypoid masses obstructing the lumen of the distal colon (b, c) in comparison to the normal colon (a).
<img src="/files/ftp_upload/4100/4100fig4.jpg" alt="Figure 4" /> 
Figure 4. Longitudinally opened mouse colon illustrating gross appearance of tumors. Note the higher tumor burden in the distal colon/rectum (left upper image), and the characteristic rugated texture of the proximal colon (right upper image) with little tumor growth. A close up view of the distal colon shows numerous tumors of varying sizes (below). 
<img src="/files/ftp_upload/4100/4100fig5.jpg" alt="Figure 5" /> 
Figure 5. Tumors highlighted by application of Alcian blue stain. Note how the dye emphasizes the normal texture of the colon as well as the borders of each individual tumor. Such staining can be helpful in the precise measurement of tumor areas by ruler or digital measurement. 
<img src="/files/ftp_upload/4100/4100fig6.jpg" alt="Figure 6" /> 
Figure 6. Representative distribution of the average number of tumors per mouse treated with AOM/DSS. Note the majority of tumors are located in the distal colon and are &lt;2 mm in size. 
<img src="/files/ftp_upload/4100/4100fig7.jpg" alt="Figure 8" /> 
Figure 7. Paraffin-embedded longitudinal sections of colon in cassette (above) and on slide following H&amp;E stain (below). Note that residual Alcian blue stain does not interfere with H&amp;E staining. A large tumor is circled on the slide image (circled). The designations "distal," "middle," and "proximal" result from sectioning the entire colon in thirds between the cecum and anus.
<img src="/files/ftp_upload/4100/4100fig8.jpg" alt="Figure 8" /> 
Figure 8. Representative histology of a tumor resulting from AOM/DSS administration in the distal colon. H&amp;E, BrdU, and &beta;-catenin stained slides at 50X (Top panel) and 400X (Bottom panel) respectively demonstrate dysplastic changes similar to human adenocarcinomas of the colon.

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No conflicts of interest declared.

Treatment of mice with AOM and DSS rapidly and effectively models human colitis-associated cancer. Hypotheses regarding heritable factors contributing to colitis-associated cancers can be easily studied with genetically engineered mice.13, 16 Alternatively, the effect of pharmacologic targets in colitis-associated cancer can be studied by employing wild-type mice.
While this model is highly valued by those interested in the study of colon tumor development in the setting of inflamma...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td> C57BL/6J Mice</td>
 <td>Jackson Laboratory</td>
 <td>000664</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Azoxymethane (AOM)</td>
 <td>Sigma Aldrich</td>
 <td>A5486-100MG</td>
 <td>Stock solution: dilute to 10 mg/ml in distilled water to be kept at -20 &deg;C as 0.5 - 1 ml aliquots.
 
 Working solution: dilute stock to 1 mg/ml in isotonic (0.9%) saline</td>
 </tr>
 <tr>
 <td>Dextran Sulfate Sodium (DSS)</td>
 <td>TdB Consultancy </td>
 <td>DB001</td>
 <td>MW 40 kDa (36-50 kDa preparations from other sources are acceptable; The same lot should be used for a single experiment)6</td>
 </tr>
 <tr>
 <td>Coloview miniendoscopic system</td>
 <td>Karl Storz </td>
 <td>Multiple</td>
 <td>See Becker et al. for detailed explanation of equipment and setup.11</td>
 </tr>
 <tr>
 <td>TPP Rapid FILTERMAX 500 ml 
 Bottle-Filter, 0.22 &mu;m PES</td>
 <td>Midwest Scientific</td>
 <td>TP99500</td>
 <td>Any standard tissue culture filter is acceptable</td>
 </tr>
 <tr>
 <td>Ethyl Alcohol 200 Proof ASC/USP</td>
 <td>Pharmaco-AAPER (or other)</td>
 <td>11ACS200</td>
 <td>Dilute to 70% in distilled water</td>
 </tr>
 <tr>
 <td>Isoflurane, USP</td>
 <td>Butler Animal Health Supply</td>
 <td>4029405</td>
 <td>Place mouse in glass jar with gauze or a small cloth soaked in anesthetic</td>
 </tr>
 <tr>
 <td>18G Straight Gavage Needle</td>
 <td>Braintree Scientific</td>
 <td>N-008</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphate Buffered Saline (PBS)</td>
 <td>Sigma Aldrich</td>
 <td>P5493</td>
 <td>Dilute to 1X (0.01 M) in distilled water</td>
 </tr>
 <tr>
 <td>Cold Tray (Tissue Tek II Cold Plate)</td>
 <td>Fisher Scientific</td>
 <td>NC9491941</td>
 <td>Store at -20 &deg;C</td>
 </tr>
 <tr>
 <td>ImageJ Software</td>
 <td>NIH (free download)</td>
 <td>&nbsp;</td>
 <td><a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a></td>
 </tr>
 <tr>
 <td>Formaldehyde (37%)</td>
 <td>Fisher Scientific</td>
 <td>F79-500</td>
 <td>Dilute to 10% in PBS</td>
 </tr>
 <tr>
 <td>BD Bacto Agar</td>
 <td>Fisher Scientific</td>
 <td>DF0140-01-0</td>
 <td>Use hotplate to create 2% solution in distilled water</td>
 </tr>
 <tr>
 <td>Miltex Eye Dressing Forceps</td>
 <td>MedPlus Inc.</td>
 <td>18-780</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Miltex Eye Scissors</td>
 <td>MedPlus Inc.</td>
 <td>18-1430</td>
 <td>Curved points prevent damage to colon during opening.</td>
 </tr>
 <tr>
 <td>Alcian Blue 8GX (powder)</td>
 <td>Sigma Aldrich</td>
 <td>A5268</td>
 <td>Add 1 g powder to 100 ml 3% acetic acid (3 ml glacial acetic acid + 97 ml distilled water)</td>
 </tr>
 <tr>
 <td>1 mL Tuberculin syringe with attached 26 G x 3/8 in intradermal bevel needle</td>
 <td>BD</td>
 <td>305946</td>
 <td>For injection of AOM</td>
 </tr>
 </tbody>
</table>

modeling colitis-associated cancer with azoxymethane (aom) and dextran sulfate sodium (dss)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Watch this Scientific Journal Video about Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS at JoVE.com

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

modeling-colitis-associated-cancer-with-azoxymethane-aom-dextran

Research

JoVE Journal

Medicine

46.6K 次观看.  Washington University School of Medicine. 我们展示了一个协议，氧化偶氮甲烷（AOM）的遗传毒性剂，其中给药后由促炎性剂硫酸葡聚糖钠（DSS）的迅速的三个周期的和一贯生成结肠肿瘤在小鼠与在人类结肠炎所见的形态学和分子的相似之处相关的癌症。

视频: Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...

Modeling and Simulations of Olfactory Drug Delivery with Passive and Active Controls of Nasally Inhaled Pharmaceutical Aerosols

There are many advantages of direct nose-to-brain drug delivery in the treatment of neurological disorders. However, its application is limited by the extremely low delivery efficiency (&#60; 1%) to the olfactory mucosa that directly connects the brain. It is crucial to develop novel techniques to deliver neurological medications more effectively to the olfactory region. The objective of this study is to develop a numerical platform to simulate and improve intranasal olfactory drug delivery. A coupled image-CFD method was presented that synthetized the image-based model development, quality meshing, fluid simulation, and magnetic particle tracking. With this method, performances of three intranasal delivery protocols were numerically assessed and compared. Influences of breathing maneuvers, magnet layout, magnetic field strength, drug release position, and particle size on the olfactory dosage were also numerically studied.
From the simulations, we found that clinically significant olfactory dosage (up to 45%) were feasible using the combination of magnet layout and selective drug release. A 64 -fold higher delivery of dosage was predicted in the case with magnetophoretic guidance compared to the case without it. However, precise guidance of nasally inhaled aerosols to the olfactory region remains challenging due to the unstable nature of magnetophoresis, as well as the high sensitivity of olfactory dosage to patient-, device-, and particle-related factors.

This manuscript reviews the modeling and simulations of different protocols to deliver medications to the olfactory region in image-based nasal airway models. Multiple software modules are used to develop the anatomically accurate nose model, generate computational mesh, simulate nasal airflows, and predict particle deposition at the olfactory region.

There are many advantages of direct nose-to-brain drug delivery in the treatment of neurological disorders. However, its application is limited by the ...